Type 1 diabetes (T1D) is an autoimmune disease characterized by T cell-mediated destruction of the pancreatic islet beta cells. In the NOD mouse model of the disease, CD8+ T cells are required pathogenic effectors that have begun to show promise as therapeutic targets. CD8+ T cells specific for beta cell antigens are also present in the peripheral blood of T1D patients. We are working to harness the potent tolerogenic properties of dendritic cells (DC) to develop an immunotherapeutic intervention for T1D that targets CD8+ T cells. We have found that antigen targeting to steady-state DC via the DC endocytic receptor DEC-205 can lead to T cell tolerance even in the context of ongoing autoimmunity in NOD mice with known tolerance defects. Transition of this strategy to the human disease will require a better understanding of its therapeutic efficacy and the factors that determine its outcome. Antigen delivery reagents appropriate for human use, and preclinical models suitable for their testing, will also need to be developed and evaluated, and the identification of antigenic targets relevant to large numbers of patients will also be required. These needs will all be met upon completion of the Specific Aims proposed in this renewal application. In Aim 1, using both murine and human T cells, we will test the hypothesis that T cell receptor (TCR) affinity and/or prior antigen experience influence the outcome of exposure to an antigen delivered to steady-state DC via anti-DEC-205, an outcome that can range from T cell deletion, non-responsiveness, or development of a regulatory phenotype. These studies will be conducted using sets of TCRs having defined and variable affinities. In Aim 2, we will test the hypothesis that simultaneous delivery of multiple critical CD8+ T cell epitopes to DC via anti-DEC-205 will lead to improvement of disease course in NOD mice and in NOD.m2m-/-.HHD(HLA-A2).hDEC-205 mice to be developed here. In Aim 3, we will examine the HLA-restricted CD8+ T cell response to the important beta cell antigens IGRP and insulin using islet-infiltrating cells from NOD mice transgenic for one of four HLA molecules that we will additionally engineer here to express human IGRP or insulin. As each HLA molecule to be examined is representative of a unique HLA supertype, and a peptide identified using one supertype member will often also bind other members of that supertype, our strategy to identify epitopes using our panel of HLA- transgenic mice expressing human beta cell antigens should result in monitoring and therapeutic reagents relevant to large numbers of patients and at-risk individuals. Our proposed studies will have important implications for the development of antigen-specific therapeutics for T1D and will provide information that will help to guide the future development of immunomodulatory therapies for this disease.